JPS60203195A - Development of plant gene - Google Patents

Abstract

There is provided a DNA shuttle vector comprising T-DNA having inserted therein a plant gene comprising a plant promoter and a plant structural gene, the plant promoter being adjacent to the 5 min -end of the plant structural gene and the plant structural gene being downstream from the plant promoter in the direction of transcription.

Description

[Detailed description of the invention] (Technical field) The present invention relates to the expression of plant genes.

(Prior art) Shuttle vector Ruvkun and Yasubel (1981) Nat
ure 289:85-88°, allows for the insertion of foreign genetic material into selected locations in large plasmids, viruses, or genomes. There are two major issues when dealing with large plasmids or genomes. One is a large plasmid with many sites for each restriction enzyme. Certain site-specific cleavage reactions are not reproducible, and multi-site cleavage reactions followed by ligation pose major difficulties as they perturb the order and orientation of many fragments that are not desired to be altered. Second, the transformation efficiency using large DNA plasmids is very low. Shuttle vectors can overcome these difficulties by easily inserting foreign genetic material into a small plasmid, often in vitro, and then transferring it to a large plasmid, usually by in vivo techniques. ~Ruhekku consists of one common plasmid, DN8, which can be introduced into the ultimate recipient bacterium.

It also contains a copy of the fragment of the recipient genome into which foreign genetic material can be inserted, and a DNA segment encoding a selection trait that is also inserted into the recipient genome fragment. Selectable traits ("markers") are easily inserted by transboson mutation +1'4 induction or restriction enzyme gauze.

The human vector is a trivalent hybrid (Ru v k
un and eightusubel, supra), direct transfer of self-mobilizing hekucu in biamidation 5:14, direct uptake by Agrobacterium cells into the external DN t7J (M, l1
olsLerset, at, (197B) Mole
c, Gen, Genet, 163:181-18
"Transformation" using the conditions of 7), spheroplast fusion of Agrobacterium with other bacterial cells.

It is introduced by uptake of liposome-encapsulated DNA or by infection of a shuttle vector on a virus that can be packaged in vitro. A triadic cross involves a cross between a strain carrying a mobile plasmid containing genes for plasmid mobilization and conjugative transfer and a strain carrying a shuttle vector. If the shuttle hector is transferable by a plasmid gene, it is transferred to a recipient cell carrying a large genome, such as the Ti or Ri plasmid of an Agrobacterium strain.

After the shuttle vector has been introduced into the recipient cells.

Double crossover with one recombination on either side of the marker is expected. This phenomenon is caused by the DN containing the marker.
Transfer the A segment to the recipient genome.

In order to select for cells deficient in a single sha1-lehekku that would replace the insert with a homologous segment lacking the insert, the shuttle vector is either incapable of replication in the ultimate recipient cell or It must be compatible with existing independently selectable plasmids in the cell. One common means to do this is to equip the third parent with another plasmid that is incompatible with the shuttle lid and has other drug resistance markers. Therefore, if you select both drug stiffness.

Surviving cells are only those in which the marker of the shuttle vector has recombined with the recipient genome.

If the shuttle vector has extra markers, select and eliminate cells that have the complete shuttle vector integrated into the recipient plasmid, resulting in a single transfer between shuttle vector ζ recipient plasmids. can. If the foreign genetic material is inserted within or in close proximity to the marker to be selected, it will be integrated into the recipient plasmid as a result of the same double recombination. If the foreign genetic material is inserted far from the marker, rather than within or close to the marker in a homologous fragment, recombination may occur between the foreign genetic material and the marker, making it impossible to transfer the foreign genetic material. Dew.

Shuttle vectors have proven useful for manipulating Agrobacterium plasmids.

References D, J, Garfinkel et Dan, (19
81) Ce1l 27:143-153. Eight, J
, M., Matzke and M., D., Chilton.
(1981) Molec, J., Appl, Genet.
, top: 39-49. and J. Leemans Dandan,
(1981) Malec, J., Appl, Ge.
net, top: 149-164, where the shuttle vector is referred to as an “intermediate vector”.
ctors)''.

Agrobacterium - Overview Gram-negative bacteria, belonging to the genus Agrobacterium of the family Rhizobium, include Agrobacterium tumefaciens (A,
tumefaciens) species and Agrobacterium
There are A. rhizogenes species. These species are responsible for plant crown gall disease 2 hairy root disease (h
airly root disease). Crown gall is characterized by the transformation of undifferentiated tissue into a gall. Hair roots are teratomas characterized by abnormal root induction in infected tissue. In both diseases, the abnormally proliferating plant tissue produces one or more amino acid derivatives, commonly known as obine, that are not normally produced by the plant.

This is catabolized by the infecting bacteria. The known obines are classified into three families, the typical members of which are octopine, tubarine, and agropine. Cells of Tatsumi constantly proliferating tissues can be propagated in culture and, under appropriate conditions, can be regenerated into complete plants while maintaining the transformed phenotype.

In Agrobacterium tumefaciens, Ti (tumor-inducing A)
￥ ; Tumor-inducing) plasmid,
In Agrobacterium rhizogenes, Ri (root R
A4; has a large plasmid called rooL-inducir+g) plasmid. When these plasmids are eliminated from bacteria, they lose their pathogenicity. Ti plasmid is T
- called DNA (transfer DNA). In tumors, it is integrated into the genome of the host plant. Contains areas. T-'
DNA encodes several types of transcripts. Mutational studies have shown that some of these are involved in the induction of 1ljl+ tumor growth. tn+l, tn
+r and tm4 gene mutations each cause giant tumors (in tobacco).

Indicates root appearance tendency and shoot inducing tendency. T-DN
A also encodes at least one opine synthetase gene, and the Ti plasmid is often.

They are classified according to the opines with which they can be synthesized. Each T-D
The NA gene is under the control of the T-DNA promoter.

This T-DNA promoter is structurally similar to eukaryotic promoters and appears to function only in transformed plant cells. Ti plasmids also carry genes outside the T-DNA region. These genes are obine catabolic 1
Involved in oncogenicity, agelodin sensitivity, replication, and autotransport into bacterial cells. The Ri plasmid has a similar structure to the Ti plasmid. The series of genes and DNA sequences involved in the transformation of plant cells are hereinafter collectively referred to as transformation-inducing factors (TIPs).

The name TIP therefore encompasses both Ti and lli plasmids. The captured segment of TIP.

Here, it is referred to as T-DNA, and is referred to as Ti plasmid or R
i plasmid. Recent general review of diseases caused by 7Globacterium Hari, J., Marlo (1982)
).

8dv, Plant Pathol, 1:139-
178. L, Shi1. Ream and M, P, Gor.
don (1982), 5science 218
: 854-859. and Gate, W., Bevan an.
d M, D, Cbilton (1982), 8nn.
.

Reb, Genet, 16: 351-384:
G, Kahl and J, Sct+ell (198
2) Molecular Ba1olo of Pl
Ant Tumors.

Agrobacterium - Plant cells can be transformed by Agrobacterium by a number of known methods; for example, co-cultivation of plant cells with Agrobacterium; direct infection of plants; plant protoplasts and Agrobacterium; fusion of umspheroplasts; direct transformation by uptake of mMtlNA by plant cell protoplasts; complete bacterial transformation of protoplasts with partial cell wall regeneration; T-DNA of protoplasts
Transformation with containing liposomes; use of viruses carrying T-DNA; microinjection, etc. Any method is sufficient as long as it ensures that the gene is expressed and stably transmitted through mitosis and meiosis.

Infection of plant tissues with Agrobacterium is a simple technique known to the skilled technician. (ExampleCu1ture
Methods for Plant Pathol
ogists, eds,: D.S., Ingramms.
and J, P, I (e1gesor+, pp, 20
3-208). Plants can be damaged in any of a variety of ways, such as cutting with a knife. Make a hole with a needle. Or rub it with an abrasive. The wound is then infected with a solution containing tumor-inducing bacteria. Other methods of infecting whole plants are potato tuber pieces (D, Anand and G).
, T., 1Ierberlein (1977) Ame
64:153-158) or a small piece of tissue, such as a tobacco stem fragment (Binns et al., ). RB 1m.

Thereafter, tumors can be tissue cultured in medium without plant hormones. Hormone-independent growth is typical of transformed plant tissues, and culture! This contrasts greatly with the normal conditions for the growth of IJl tissues (8, C. Braun (1956) Ca
Ncer Res, 16:53-56).

Agrobacterium is also used in cell and cultured cells (Marton et al. (1979)).
re η1 no 129-131) and jllt mesophyll protoplasts of tahini can be infected. In the latter technique, Agrobacterium cells were then added to the culture to allow time for some new cell wall regeneration and then killed by addition of antibiotics. Only cells that were in contact with Agrobacterium teumefaciens cells and retained the Ti plasmid formed callus when plated on a hormone-free medium. Most calli had enzymatic activities related to opine assimilation. Other researchers (R, B, IIorsc
h and R, T, Fraley (18January
ry1983 15th Miami Winter
Symposium) was co-cultivated to obtain calli that were transformed and proliferated in a hormone-independent manner at a high frequency (10% or more), and 95% of the calli produced opine. M, R, D
avey at Dan, (1980) in Ingram
and Lelgeson, supra, pp. 209.
-219. describe the infection of old cells regenerated from protoplasts.

Plant protoplasts can be transformed by direct uptake of the TIP plasmid. M.R.Davey et al.
, (1980) Plant Sci, Lett, 1
8:307-313. and M, R, Davey e.
tal, (1980) in Ingram an
d Ilgeson, supra, transformed petunia protoplasts with a Ti plasmid in the presence of poly-L-α-ornithine and cultured them to show a phenotype of obine synthesis and hormone-independent growth. It was subsequently shown that polyethylene glycol promotes Ti uptake and that certain T-DNA sequences are incorporated into the genome. (J, Dra
per et al. (1982) Plant an
dFujiwara+pp, 515-516) o
F., A., Krens et al.

(1982) Nature 296: 72-74
reported similar results using polyethylene glycol followed by calcium shock, but in their results the incorporated T-DNA contained contiguous portions of Ti plasmid sequences.

Another method for uptake of DNA is the use of liposomes. Preparation of DNA-containing liposomes was performed using Papahad.
Jopou Ios U.S. Pat. No. 4,078,05
No. 2 and No. 4.235,871. A method of introducing Ti-DNA using liposomes has been reported (T,
NaBa taeta+, (1982) in Fu
jiwara, supra, pp. 509-510 and T.
, Nagata (19131) Mol, Gen, G.
enet, 184: 161-165). 11(
The following system involves the fusion of plant and bacterial cells with their cell walls removed. An example of this technique is S. Ilasezawaet a.
l, (1981) Mo1. Gen, Genet,
1B2: 206-210, transformation of periwinkle (Vinca) by spheroplasts of Papilio bacterium. Plant protoplasts can take up Agrobacterium cells with incomplete cell walls. (S,llasezawa
et al. (1982) 1nFuj iwara+
Ibid., pp. 517-518).

i”-DNA is transferred to the regenerated tissue resulting from the fusion of two protoplasts, and only one is transformed (G
, J.Wullems at Dan, (1980) Th
eor, 8 ppl.

Genet, 56: 203-208), as detailed in the section on plant reproduction, T-DNA is also passed through meiosis and transmitted to progeny according to simple Mendelian rules.

Agrobacterium - Plant Regeneration Differentiated plant tissues with normal morphology were obtained from crown gall tumors. A, C, Braun and 11. '
NyWood (1976) Proc, Na11. 8c
ad, Sci, USA 73: 496-500°, grafted tobacco malformed species (teraLomas) onto normal plants and obtained normal-looking shoots capable of flowering.

When this shoot is placed in a culture medium, it has the ability to produce opines.

It retained plant hormone-independent growth ability. In selected plants, these tumor phenotypes do not appear to be transmitted to progeny;
It probably disappeared during meiosis. (1 n.

Turgeon et Dan, (1976) Proc.
Natl, Acad, Sci.

US Pass 73: 3562-3564>. It spontaneously lost its tumor properties. Alternatively, plants arising from seeds of a malformed species initially appeared to have lost all T-DNA (F, -M, Yar+BeL Dan, (1980) I
n Vitro 16: 87-92+ F, Ya
ng et al. (1980) Molec, Gen.
, Genet.

177 Near 07-714. M cmmars cL
al. (1980), 1°Mo1. Biol, 1
44: 353-376). However, hormones (1
In subsequent studies using plants that reverted after treatment (■/l kinetin), plants that underwent meiosis showed that T
-DNA gene was lost, but homologous sequences were maintained at both ends of T-DNA. (F, Yang
and R, Il, Simpson (1981) P
roc.

Natl, 8cad, Sci, USA 78: 41
51-4155), G, J.

Wulleg15 B (Dan, (1981) Ce1l
24: 719-724. Furthermore, genes related to opine assimilation are transmitted through meiosis, although the plant is male sterile.

It was also shown that T-DNA can probably be inherited according to Mendel's law. (G, IQ (111em
Rainbow, (1982) in A, Fujiwar
a, supra). 1.0ttenat al, (198
1) Mo1ec, Gen, Genet, 18s 3:
209-213° is a Tn7) transboson-induced Tn7 in the gene wall that produces shoots (shoot induction).
i plasmid mutations were used. When these shoes 1~ were regenerated in plants, they produced self-fertile flowers. Plants from which the epiphytic seeds germinated contained T-DNA and produced opine. Similar experiments using root-induced mutations showed that the entire T-DN8 was transmitted to offspring through meiosis.

It was shown that the tuberin gene was expressed to varying degrees among these progeny, and that the yeast alcohol dehydrogenase I gene, which had been transferred at the same time, was not expressed.
(K, 8, Barton et al, (1983)C
ell 32:1033-1043), T-DN
The regenerated tissue lacking the A sequence is probably the descendant of non-transformed cells that were present in <l1liW. (G, Oom
stiL al.

(1982) Ce1l 30: 589-597
), it was shown that roots resulting from transformation with Agrobacterium rhizogenes regenerate into seedlings relatively easily.

432-434).

Agrohatatherium - Tll'Plasmid"
Approximately half a dozen octobin plasmids were identified in the T-DNA of the TIP plasmid.
- DNA transcripts were mapped (S, B, Gel
ν1net al, (1982) Proc, Nat
l, 8cad, Sci, USA 79 Near 6-80.
L, ls111m1tzer et at, (198
2) EMrlOJ.

1: 139-146 >, and some functions were clarified (J, 'Leemans et Dan, (1
982) IiM[lOJ,±:147-152).

The four genes of the octopine-type plasmid are tms, t
This was well defined by 1-trans boson mutagenesis involving mr and dan. (D.J.

Ti plasmids with mutations in these genes stimulate the tumor callus of Nicotinia tahacum to produce shoots,
root and become larger than normal. In other hosts, mutations in these genes can induce different phenotypes (ChilLon, M.D., Rev.

Genet, (1982)). The ``Kake'' and ``Kake'' phenotypes are correlated with differences in the levels of plant hormones present in the tumors. The difference in zytokinin:auxin ratio is similar to that seen when shoot or root formation is induced in non-transformed callus tissue in culture. (D, E.

^kiyoshi et al. (1983) Pr
oc, NaLl, 8cad, Sci.

By USA: 407-411). T-DNA with functional genes for either Difference or A (although not only for A41) can stimulate overt tumor growth. Shoot and root stimulation are promoted and inhibited, respectively, by function υ. (L, W, Ream etal,
(1983) Proc, NaLl, 8cad, Sc
i, USA 80: 1660-1664). T-
Mutations in DNA genes do not seem to affect the insertion of F-DNA into the plant genome (J, Leemans
et al. (1982) Ibid., L.W.

Ream et al. (1983) ibid). OCS genes encoding octopine synthetase are Il, De
Greve et al. (1982) J, Mo1
．． The base sequence was determined by 8 ppl, Genet, top: 499-511°. It does not have introns (intervening sequences commonly found in eukaryotic genes that are removed from the Menger precursor during post-transcriptional processing of the mRNA). Also, the eukaryotic transcription signal (TA”F
There is a water box and a polyadenylation site. Since plant cells with octopine synthetase detoxify homoarginine, the OCS gene would be an effective selection marker for plant cells transformed with foreign DNA.

(G, M, S, Van Slogteren et Dan, (1982) PlantMol, Biol, supra:
133-142). The Tubarin Ti plasmid encodes the nopaline synthase gene (Lice),
A, Depicker et al.

(19B2) J, Mo1. to pp1. Genet, top: 561-573. The sequence was determined by OC3
Like genes, there are no introns. There are two potential polyadenylation sites and a sequence that could be a TATA box.

In contrast, there is a sequence known as a CAT box, which appears to be a transcriptional signal, upstream of the CAT box. J.

C., McPherson et al. (1980)
Proc, Natl, Acad.

Sci, tlSfl 1 muff: 2666-2670
．． reported in vitro and in vitro translation of mRNA encoded by T-DNA of crown gall tissue.

Transcription of hair root T-DNA was also detected (L.

Kaii+1m1tzer eL Niji, (1982) Mo
1. Gcn, GeneL, 186:16-22).

Functionally, hair root syndrome appears to be equivalent to crown gall tumors caused by Kono's mutated Ti plasmid. (F, F, Wl+ite and E.

W, Ne5ter (1980) J, Bacter
iol, January 1:'710-720).

In eukaryotes, DNA methylation (particularly of cytosine residues) is correlated with transcriptional inactivation.

Genes that are relatively less methylated are transcribed into mRNA. Gevin et al. (1983) Nu
cleic Ac1dsRes, ±: 159-174
．． found that at least one unmethylated copy of T-DNA in crown gall tumors is always present. Many other T − in which the same genome is methylated
Containing DNA copies means one or more excess T-
Suggests that the DNA copy is biologically inactive (G.

Ooms et al. (1982) Ce1l 30
:589-597).

The Ti plasmid is outside the T-DNA region.

It coats other genes necessary for the infection process.

(M, IIolster for Tubarin plasmid
Dan Al, (1980) Plasmid 3: 212-
230. For octopine plasmids, see 11. De
Greve et al.

(1981) Plasmid 6: 235-248+D
, J., Garfinkeland E., W., Ne5te.
r (]980) J, Bacteriol 144
: 732-743, and G, Ooms (1980
) J, Bacteriol 144:82-91). The most important gene is the Ichigengo gene, which, when mutated, causes the Ti plasmid to lose its oncogenic properties.

(These loci are known as virulence-related reengineering). ! The gene acts in trans and can cause the transformation of plant cells with T-DNA that is physically located on another plasmid in a different plasmid type. (J, lille old ee et tan, (198
2) J, Bacteriol 150: 327-
331. M,-D, Chilton (18January
ry 1983) 15th Miami WinLe
r Symp,). Tubarin TiDNA appears to be involved in excision from the Ti plasmid or incorporation into the host genome, and consists of an approximately 25 base pair forward repeat sequence (d
direct repeat ) with (N,'S.

Yadav et al. (19B2) Proc.
Natl, cad, sci, tlsA neck: 6322
-6326), and the similar sequence is Octopin T
- Found adjacent to the DNA border (R
, B., Simpson et al. (1982) C.
e1l 29:1005-1014). Opine assimilation is characterized by octopine and tubalin-type plasmids, respectively. The Ti plasmid also encodes functions necessary for its own propagation, including an origin of replication. Ti plasmid transcripts are S, B, Ge1
Vin at Dan, (1981) Plasmid 6: 17
-29. found in Agrobacterium tumefaciens cells that T-DNA regions are weakly transcribed along non-T-DNA sequences. Properties controlled by Ti plasmid are Merl
o.

Above (see especially Table 2) and Ream and Go
rdon.

It is summarized by the above.

Agrobacterium-TIP plasmid DNA DNA hybridization (T, C,
Currier and E, W, Ne5ter (1
976) J, Bacteriol, 126:157
-165) or restriction enzyme analysis (D, 5ciaky
et Dan, (1978) on plasmid = 238-253
) was investigated. Tubarin-type Ti plasmids are at least 67% homologous to each other (Currier
and Netert, supra), the various Ri plasmids were found to be highly homologous to each other (P, Cotantino et al., (1981).
) Plasmid 5: 170-182) o N, I
l, Drummond and M, -D.

Chilton (1978) J, Bacterio.
I, 136: 1178-1183.

showed that octopine and tubalin-type Ti plasmids have relatively narrow homology to each other.

These homologies have been described by G. Engler et al. (19
81) J, Mo1Bio1.352: 183-2
It was mapped in detail in 2008.

They further subdivide three of the four similar regions into three (spanning the T-DNA), four (containing several "distant" trochanters), and nine similar regions (having several "distant" trochanters). I found out that it can be done. The connecting homologous regions include at least the trafi gene (involved in conjugative transfer of the T trochanter lasmid to other bacterial cells) and the trafi gene for replication and incompatibility. This region is homologous to the Snake plasmid (involved in symbiotic nitrogen fixation) isolated from a species of Rhizobium, another genus of the Rhinophyllidae (11., Prakash, (1982) Plasmid 7♂
271-280). Although the order of the four regions is not preserved, they are all arranged in the same direction. T-DNA
Part of the sequence is extremely well conserved between the tuberin and octopine plasmids (M, -ri, CI+1lt
on et at.

(1978) Nature 2: 147-149
．． ^, Depicker at Dan, (1978) N
ture 275: 150-153), R
i plasmids have between them 3 and octopine (F, F
, 11hiteand E, In, Ne5ter (1
980) J, BacLeriol, 144:7
10-720) and Tubarin (G, 1n1suleo)
et al. (1982) Plasmid 7: 45-
51') and both Ti plasmids. That region is primarily the one that encodes the "extended trochanter." Ri T DNA is the T of both types of Ti plasmid.
-1) -Although it is weak against NA.

There is considerable homology. (L, Willmitzer e
tal.

(1982) Mo1. Gen, Genet, 186
: 3]93-3197). The plant DNA of uninfected Nicotinia glauca is cT-DNA < m cell T-
It contains a sequence called Ri T-DN
It has some homology to A. (F, F, White e
tal, (1983) Nature 301:3
48-350>.

Ti (M, -D, Chilton et at, (1
977) Ce1l 11:263-271) or I? i (M, -D, C++1lLon (1982
) N, 1ture295: 432-434. F
, F. White et Dan, (1982) Pro
c.

Na11. 8cad, Sci, USA 79: 31
93-3197. L, Willmitzer (19B
2) Mo1. Gen, Genet, 1136:
16-22) A portion of the plasmid is present in tumor plant cells.
Found in NA.

The transferred DNA is known as T-DNA.

T-DNA is present in the nucleus (M, P, Nuti et al.
1980) Plant Sci, Lett, 18:
1-6, L, Willmitzer et406
0-4064) host DNA' (M, F, Tho
mashow et458-461).

M, F, TI+omashow eL al, (198
0) Proc, Natl.

8cad, Sci, USA 77: 6448-6
452. and M,F.

Thomashow et al. (1980) Ce
1l 19: 729-739° is octopine type Ti
The T-DNA of the plasmid is T-DNA on the left and right side of the T-DNA, respectively.
It was found that L-DNA and TR-DNA were incorporated into two separate locations. The copy number of TR and TL can vary (D, J, Merlo et al, (19
80) Molec, Gen, Genet.

177:637-643), the core to T-DN.

Highly homologous to Tubarin 1”-DNA (Chi
'1ton eLat, (197B), supra, and Depicker eL Dan.

(1978) supra), is required for tumor maintenance, is found in 1 L, is generally present at one copy per cell, and is required for tumor maintenance.
and “1” encodes a gene. On the other hand, TR has a large number of copies (D, J, Merlo et al, (1
980) supra) is completely unnecessary. (M, De
Beuckel-eer et al. (1981)
Molec, Gen. Qenet, 183:283
-288, 'G, Ooms 'eL Dan, (198
2) Ce1l 30: 589-597). G
, Ooms et al, '(1982) Plasmid 1:15-29, Agrobacterium tumefaciens remains virulent even when 1'R is deleted from the Ti plasmid, but 'I' remains virulent. R is T −D
It is assumed that it is involved in NA uptake. G, Ooms
et al, (1982) Ce1l: 589-
597, T-DNA is specifically deleted after being incorporated into the plant genome, but it is generally stable;
It has been shown that l1ffi tumors, which contain mixed cells with different DNA compositions, are the result of multiple transformation events. ocs exists in the TL. However, it can be deleted from the plant genome without loss of phenotypes associated with tumor growth.

The left end of the embedded TL is a forward or backward repeating T.
- consists of a DNA sequence. (R, B, Simpson
tal, (19B2) Ce1l 29:1005-
1014).

In contrast to the situation in octopine-type tumors, tubalin1'-
DNA is integrated into the host genome in a series of fragments. (M. Emmers et al. (1980)
) J.

Mo1. Biol, 144: 353-376+
P, Zambryski eLdan, (1980) 5c
ience 209:1385-1391), forward repeat sequences were observed. The T-DNA of plants generated from malformed species has a slight modification in the end fragment of the inserted DNA ([, emmers e+yudan, ・
(cited above). Analysis of the sequence at the junction between the right and left ends revealed many forward repeating sequences and one inverted repeating sequence. The latter spans the junction (Zambrys
ki et ++1. (1980) supra). The left junction should vary by at least 70 base pairs (bp).

On the other hand, the right junction is a variation of lbp (P.

Zambryski et al. (1982) J
, Molec, 8pp1. GeneL.

1:3 (if-370). The left and right ends of the junction of the repeat sequence are separated by a spacer of 130 bp or more. The origin of this spacer is unknown.

T-1) Contains an NA sequence. T-DNA is integrated into both repetitive sequences and low copy number host sequences.

N, S, Yadav et al. (1982) P
roc, Natl, 8cad.

Sci, USA 79: 6322-6326. found a site in the Tubarin Ti plasmid just outside the left end of the r'-DNA, which increases general recombination in the surrounding DNA 10 Kbp apart in bacteriophage λ. R, B, Simpson eL
al. (1982) Ce1l 29:1005~
1014. did not find any sequence within Octopine Ti gelasmi 1, but cannot rule out the possibility that it exists outside the region where the sequence was determined. The significance of "" in the Ti plasmid is unknown. If chi had a function, it would probably be used in Agrobacterium cells rather than in plants, since chi is not in T-DNA.

Manipulation of the Agrocyclium-TIP plasmid as detailed in the Shuttle Hector section.

Techniques have been developed to introduce altered DNA sequences into the desired location of the i'IP plasmid. Transbosons are easily inserted with this technique. (D.J.

Garfinkel et Dan, (1981) Ce1
l 27:143-153).

J,-P,1lernalsteen et al.
1980) Nature 287: 654-656
．． showed that a DNA sequence (here a bacterial transboson) inserted into the T=DNA of the Ti plasmid was transferred and incorporated into the recipient plant genome. Many foreign DNAs of various origins have been inserted, but until now the gene wanted to be expressed under the control of its own promoter. These genes include yeast alcohol dehydrogenase (^dh
) (K, A.

Barton eL, Dan, (1983) ),) maize 8dhl (J, Bennetzen'+unpublished) and zein, mammalian interferon and globin, mammalian virus 5v40 (J, Scl+ell, unpublished), etc. . M, l1olsterset, Il
, (1982) Mo1. Gen, Genet, 18
5: 283-289°, after the bacterial transboson (Tn 7 ) inserted into the T-DNA is incorporated into the plant genome.

Recovered in fully functional and possibly unchanged form.

Defects can be created within the TIP plasmid in a variety of ways. Shuttle Hector can be used to introduce deletions created using standard recombinant DNA techniques.

(Cohen and Boyer US Pat.
4,237+224) aPredetermined deletions at one end can be created by misexcision of the transboson. (8゜15-29), J, ll1
lle and R,5chilperooL (19
81) Plasmid 6: 151-154. showed that double-ended deletions at predetermined positions can be created using two transbosons. This technique can also be used to construct "recombinant NA molecules" iso-vivo.

DN in which the tuberin synthetase gene encodes drug resistance used to select transformed plant cells
It was used to insert the A segment.

M, Bevan (M, D, Chilton et a
l, (18 January 1983) 15th Mi
ami Winter Symp, , and J, L, Marx (1983) 5cien
ce 219:830) and R. IIorsch.
et Dan, ((18January 1983) 1
5th Miami Winter Symp, and Marx+ (see supra), inserted the kanamycin resistance gene (neomycin phosphometransferase) of Tn5 after (under the control of) the tubalin promoter.

Its production involved transforming plant cells in culture to become resistant to analogs such as kanamycin and G418. J, Scl+ell at al, (1
8January 1983) 15th Miami
Winter Symp, (Marx.

(ibid., see also) reported a similar construction method, in which T
n7 methotrexate resistance gene (dihydroflu-
1 reductase) was ligated after the tubalin synthetase promoter. The transformed cells were methotrex-1.
It was resistant. Plant cells with octopine synthase are resistant to the toxic chemical homoarginine, and G
, M.S., VanSloHteren et al.
(1982) Plant Mo1. Biol, top:
133-142. proposed the use of this enzyme as a laundry marker.

Gate, -D, Ct+1lLon Ri□a1. (1983
), supra, 8.

Defremeu is “mini-Ti”
They reported that they had created a plasmid “Tubarin T-DN”.
There is actually one restriction enzyme cleavage site in A. A mutation lacking this site was created and jP-MI[edited] to Ml fragment 1, which contains the complete tubalin T-DNA. This fragment was inserted into pRK290 along with the kanamycin resistance gene and is maintained in Agrobacterium tumefaciens and all non-T-D'N
A plasmid was obtained lacking the A sequence. By itself,
This plasmid was unable to transform plant cells. However, when it was introduced into a G. tumefaciens strain carrying the octopine Ti plasmid, tumors that synthesized both octopine and tuberin were induced. This is due to loss of tubalin T1 plasmid function in octopine Ti.
This indicates that it was complemented by the plasmid and that the tubalin "small Ti" was capable of transforming plant cells. Chilson et al. (1983), supra, also cut small Ti with Smal and cut all i-1 except for the tubalin synthetase gene and its left and right ends.
) “fd T i” lacking NA (micro-
Ti) was created.

This small Ti was inserted into the pRK290 plasmid containing the Sma1 site and used in the same manner as the small Ti with comparable results.

Il, 1. orz et al. (1982) in
Plant Ti5sueCulture 19B2.
ed:^, Fujiwara, pp, 511-5
12. In order to maintain DNAΔNA, a TIP system was used to create a plasmid hector apparently related to hyperthermia, and the tubalin synthetase gene was used as a marker.

Phaseori 4ζ gene regulation In general, genes in higher eukaryotes are highly regulated.

Multicellular organs like plants have many differentiated tissues,
Each has a unique function that requires a unique gene product.

One such tissue is the cotyledon (cotyledon).
There is. In legumes (l+4 umes), cotyledons are storage organs for seeds that store lipids, carbohydrates, minerals, and proteins until they are needed during one germination. Phoseolus vulgaris L, (French bean, kidney bean (ki)
dny bean), dry bean (navy bean)
), mung bean (also known as CBreen bean), the major storage protein is phaseolin.

This protein consists of a small number of molecular species that are very similar and in equal amounts to each other. Phaseolin is responsible for the major nutritional value of dried beans, often accounting for more than 10% of the dry weight.

Phaseolin is highly regulated during the life cycle of Phaseolus vulgaris. This protein is produced only while the seed is developing in the pod, and its levels rise according to a genetically determined schedule of synthesis from a low detection limit until it accounts for half of the seed protein. At its peak, phaseolin synthesis reaches more than 80% of cotyledon cell protein synthesis. Other times, other times! +Jl+, phaseolin combination cannot be detected. With its importance as a global nutritional source, the regulatory mechanism of phaseolin has attracted great interest in the study of phaseolin, its properties and its regulation.

(Hereinafter in the margins) (Object of the Invention) The invention disclosed herein provides a plant having genetically modified plant cells into which a plant gene is introduced and expressed therein. Furthermore, the present invention provides a plant tissue whose genome contains an 'r-DNA, which is expressed in the plant cell. Here, a T-DN modified to contain an inserted plant gene expressible in plant cells
Provided are novel bacterial strains of bacteria of the genus Agrobacterium capable of replication, including A. Furthermore, the present invention has replication ability in E. coli (E. coli), and T-
T-D containing DNA and further contained within the plasmid
Novel plasmids are provided that contain plant genes inserted into NA.

The experimental studies disclosed herein demonstrate that plant genes are expressed in plant cells after introduction via T-DNA.

That is, this was carried out by inserting a plant gene into T-DNA by a known method and introducing the T-DNA containing the inserted material into plant cells, and we believe that this is the first example. The experiments disclosed here.

In addition, plant genes containing introns are T-1) N
We believe that this provides the first example of expression in a plant cell introduced via A. These results are 'I゛-DNA
This is surprising in light of the fact that all known genes expressed within the human body (whether T-DNA endogenous genes or inserted foreign genes) do not contain introns as far as is currently known. teeth.

In addition, when a promoter outside the T-DNA gene is inserted into the 1''-DNA by those skilled in the art.

Considering the fact that we have not been able to show a single example of it functioning to control expression in plants, this is not easy to imagine.

The present invention is useful for introducing useful plant genes from other plant species or strains to genetically modify plant tissues or whole plants. Such useful plant genes are storage proteins,
lectins, disease, insect and herbicide resistance factors,
Examples include, but are not limited to, genes for factors that confer resistance to environmental stress, as well as genes for specific fragrances.

The present invention has demonstrated that the phaseolin 1 gene, which is the main storage protein of legumes, can be used in plants such as Castor I and Taha:T.
This is exemplified by the introduction and expression of \. Once plant cells expressing T-DNA have been obtained, plant tissues and whole plants can be synthesized using methods known in the art. method, it can be regenerated from scratch. The regenerated plants are then propagated in the usual way.

U, rijinsaomi two-tone trochanteri can be transferred to other plants using normal plant improvement techniques. For example, f
It can be used for its protein content and nutritional value. Other applications of the present invention, such as the exploitation of other genes introduced into other plant species, will be readily apparent to those skilled in the art. The present invention essentially provides any plant gene with T-1)N
It can contain AOm and maintain stable replication. It can be applied to any plant species. Generally, these species include, but are not limited to: Namely, sunflower (Composi family Lcacaceae), tobacco (family Solanano S 01 a na Ce ae family), alfalfa, soybean and other legumes (family IeBum ! n03 (family !ile family) and humans:
Opposition of lji): gHQ几1:・)na (dicoLylcdonous) is a twin plant.

(Structure of the Invention) The following definitions are provided to eliminate ambiguity regarding the meaning of use in the Unknown Book and the claims and the scope of rights.

A: This term used in the NΔ upper segment language C; Isuu J, Chume Funkonins and Age 1: J
It is naturally derived from all strains of Agromycocytes, including Rhizogenes, Rhizogenes.
Contains NA.

In the case of the latter strain, previous researchers sometimes referred to it as R'-DNA. Furthermore, as used herein, the term "T'-DNA" includes any changes, modifications, mutations, insertions, and deletions that occur naturally or by laboratory manipulation. -The right and left ends of DNA are all 'l' -1) NA's 1,'I
It means that there is a sufficient amount to realize the function of stable 3.1 [embedding].

11 sisters and four children: The use of plants includes structural and regulatory elements of plant genes, i.e. elements that are foreign to the genes of I'-DNΔselfJ:I. Plant origin r-is i'-1) NA・＼Plant origin 1)
The motor (a region of the transducer that provides and can regulate the initiation of transcription and the initiation of translation) and the transcriptional trochanteric Bti are associated with each other. (areas containing or excluding one or more regions). This 1icc
! 1'?
Nhef'I: 1 may have the function of controlling the senonong; it may also include the 3° --11^II il< region.

The P1nid[-ter and the 11,1-generating element will be the same or 5° different from the existing gene [1jil],
Also same or 5r1. iσ animal source 111. For example, ii! (In the case of a biological gene, it is possible to make the motor identical to a certain plant gene, or to the co-IS region (intron presence or absence) of a single gene, or &J
In vitro 4M compositions consisting of other UI motors derived from other 1υ species may also be used. As defined herein, the coding region of the plant trochanter contains a cDNA copy of the structural gene r of a plant gene. The promoter and call region may also contain naturally or artificially induced modifications; Can include chemically synthesized segments.The coated segment may itself be JA1 Naturally occurring or synthetic, hybrid 4
BL white matter 21-t'f5j, on the origin of number [1jiK
Even if there is a mixture that does, 1, no.

% Q & 111 NI: Crown goal's] 1 root 2
- 1714 isoclasts containing assemblages of various shapes of li:4m plant cells, such as flowers (15), seeds, tumors, and liver, callus (3.11).

Or, it includes Oba, 11 weavings derived from it.

City? Production of genetically modified plants expressing plant genes introduced through DNA, culture 111f proboscis, l, and protoplasts With the technology of)
It is a combination of currently known specific knowledge and techniques-H] stages. In many instances. Alternative measures exist at each stage of the entire process. The selection of means is basically 1” I
Depending on factors such as the choice of P, the plant species to be modified and the desired regeneration method, all of which provide alternative process steps that one skilled in the art may select and use to achieve the desired result. 5. The basic features of the present invention are the properties of plant genes and fM construction.

This is the method of inserting into T-1) NA. genetically(
11 The remaining step to develop the ornamental proboscis is to transfer the NA (Itodecor"['-1') into the plant cell (
Where.

Stably integrated into plant cells as part of modified 1"-DNA or plant cell genomes), including: There is a technology related to regeneration.This technology is based on transformed plants.
The method may include steps relating to selection and detection of cells and transferring the transgene r- from the initial transformant to a commercially acceptable culture.

The basic 111 colors of the present invention are the J, l, j stains of i''-1) NA that contain the inserted plant genes defined above.

According to previous studies, these regions are 411 f unless disrupted by transfer of sequences 11 in the immediate vicinity of the plant gene-insertion site; JT-1)
! It may be any gene that is essential for the insertion of ili T-DNA into the plant genome. The preferred insertion site is the one with 1σ in the area where the insertion is most viscous), and
1. 'l' gene-1 and iI! as shown in FIG. ! Anal plate that spans figure section 13! This is the region called 1.6° in the II-f fragment. There is no expression that correlates with the latter inversion.

The term "1.6" here refers to this actively transcribed region of the T"-DNA.i"-11N
A can be obtained from any plasmid 1. Plant genes are inserted using standard techniques well known to those skilled in the art. In terms of orientation, the inserted plant gene can be inserted in any direction, and one of the two possible orientations will not serve a function.If a gene is inserted at a different position in the T-DNA, factors such as chromatin structure 1. The difference in the degree of expression of the phaseolin gene is -j'
p'l' i +590) -C1 in the Lee ↓ trochanter.
Once inserted into the site, readily detectable expression levels are reached.

A simple method to insert 11j1 proboscis into i゛-DNA.

There is the already mentioned 7-1 Torhector, which is an Escherichia 2J IJ-1) NΔ segment (i'-1) that has been incorporated into a viable plasmid.
l? ;1” segment). This 'l'
-]) The NΔ segment I~ preferably has one restriction site unique to the shuttle hector (j). The plant gene is inserted at a specific +J'I: site within the T-DNA segment. This sort is 1 hectare
l! ! [17 g] Hakusuumu strain, preferably it has a T-L)
i' -1) $ of strains homologous to N△segment 1
Transferred to cell 111. The transformed Age I-1 strain 11 was inserted into the existing segment of the Tl plasmid with the i''-DNΔ segment 1- of sorted 1 lehector (selecting the reversal phenomenon of double homology 3, 11). Article (
'l human I! Ilri(?Isaichiru.

Kogode i Mbeta Kata 1 (Chai (IM fiQ+ T
-1) NA can be transferred to plant cells by any technique known for the molecule Y. For example, this transfer can be accomplished by direct infection of a plasmid carried by a new Agrobacterium strain containing plant genes incorporated into the NA, or by coexistence of Agrobacterium strains and proboscis T, +n cells. The former technique, direct infection, eventually leads to the appearance of tumor bodies or crown galls at the site of infection. The crown gall cells are then grown in culture, and the lj'? The inserted T-DNA segment is h-
It will be regenerated into a complete plant. Coexistence 1i; Depending on the feeding method, a certain part of the plant X111 vesicles will be transformed into '1'. Immediately, the T-DNA moves into the cell and is inserted into the cell genome (16).
IQ K+: + ++ follicles must be selected or searched to distinguish them from untransformed cells. The selection is i' −
1) Easily achieved by using a selectable marker that is incorporated into the NA (along with the 11C substance).

As an example, nopaline synthetase promoter control (
・Shiva rdolph A-rate reductase or neomynon phasphotransf J-lase expressed in . These markers are selected by growth in media containing methotrexide or kanamycin or their derivatives, respectively. Furthermore, T-DNA is an endogenous marker, such as a gene or gene group that controls hormone-independent growth in Ti-induced LS III l& culture.

It has a gene or gene group that controls the abnormal morphology of the Ri-sen tumor route, and a gene group that controls resistance to toxic compounds such as amino acid analogues (the resistance is provided by opine synthetase). Search methods well known to those skilled in the art include measurement of opine production, specific hybridization to characteristic RNA or 'r-1)NΔ sequences, or ELISA (abbreviation for enzyme-linked immunosorbent assay), radioimmunoassay, and specific protein immunoassays such as "Western" plots.

Another approach to the Shuttle Hector strategy is the use of plasmids containing T-DNA or modified 'F-1) NA into which plant genes have been inserted, ie, plasmids that can replicate independently within the Agrobacterium pulva.

Recent data shows that Agrobacterium strains have T-DNA
If the plant has a trans-acting gene that has the function of promoting the movement of cells into the j1 cell.

It has been shown that the T-DNA of such a plasmid can be transferred from a Bacterium strain to plant cells. Plasmids that contain T-DNA and can replicate independently within the Agrobacterium are referred to herein as "sub-TIP" (sub-TIP) plasmids. There is a range of variation in which the "sub-TIP" plasmids differ in the "-1) NAO■ they contain. The ends of that range are the T-D from the 'I'IP plasmids.
All NA remains.

In particular, it is referred to as a "mini-TIP" (mini-TIP) plasmid. The other end of the range is “r”-1) N A
All but a minimal amount of DNA around the border is deleted. The remaining portion is the minimal form necessary to be able to transfer and integrate within the host cell. Such a plasmid is
jf5"l' I P"(micro-T11'
) is called.

The Zabugo IP plasmid has the advantage of being small and relatively easy to manipulate directly. After inserting the desired gene, T-1) can be easily transduced directly into plants containing the lance-acting gene that promotes NA transfer. Introduction into an Agrobacterium strain is conveniently accomplished by transformation of an Agrobacterium strain or conjugative transfer from donor 11tl bacterial cells, techniques well known to those skilled in the art.

Regeneration is accomplished using known techniques. The purpose of the regeneration step is to obtain a whole plant that will successfully reproduce and reproduce but retains the integrated "I'-DNA". Regeneration techniques are based on principles known in the art. According to T.
- Some variation depending on the origin of the DNA, the nature of the modifications therein, and the species of 11α product transformed. Plant cells transformed with Ri-type T-DNA are easily regenerated by known techniques without any extra experimentation.

Plant cells transformed with Ti-type T-DNA are
In one example, it can be regenerated by properly manipulating the 18-year-old Polmonreher. Preferably, however, Ti-transformed tissue is most easily regenerated if the go-DNA has undergone mutations in the Tmr and/or Tms genes. Inactivation of these genes restores the normal hormonal physiology of the transformed tissue, making it extremely easy to manipulate the pormonhormonal system of the tissue in culture, resulting in a more normal hormonal physiology that regenerates in two cycles. bring about plants with. In some cases, the tumor cells have an integrated T-DNA and express a T-DNA gene, such as novalin noncetase. And it is also possible to regenerate shoots expressing the inserted plant gene. This shoot can be maintained by vegetative cells by being grafted onto a plant with roots, and fertile flowers can be attached to it. The shoot thus becomes the parent plant for normal progeny plants that carry the T-DNA and express the plant genes inserted therein.

The transformed plants F, 11 genes of genotype I+:L are selected into cages by which their cells are capable of regeneration at an early stage of life in Inhydro's medium. If cultivars of agronomic interest are not adapted to these manipulations, more permissive changes are made first. After regeneration, a new J,r′j outpatient (
1! The idiopathic gene is readily transferred into the desired agronomic cultivar plant by techniques known to those skilled in the art of plant breeding and plant engineering. The first hybrids are obtained by crossing the transformed plants with these agronomically cultivated plants.

These hybrids can then be backcrossed with plants of the desired genetic background. Progeny are then continually searched and selected for new phenotypes as a result of the linked presence of the Nun-incorporated T-DNA or the expression of the inserted plant gene. In this way, after a number of backcrosses, plants with inserted plant genes can be produced with a genotype that is woody identical to the desired parent plant.

(Example) The following example is 'l' IP and Age l-1 Hacterium l
, etc.) are well known and accepted by those skilled in child biology and manipulation.
J makes use of many techniques that can be applied; such methods are not always well described.

Enzymes are obtained from commercial sources and used according to the advice of Hender or other variations known in the art. Reagents, buffers and media conditions are also known in the art. Reference studies on such techniques include: I1. Wu, CD (1979) Meth,
lEnzymol, ρ8; J, Il, old 11er
(1972) [experiments in Mo.
1ecular Genetics; R, Davi
s Dandan, (1980) group! 13 cicadas"↓G Gaihei L Shito Il, F, 5chleif and l', c,
Wnesink (1982) 1'ractical
Methods in Mo1ec+(↓;rr Bi
- With the exception of plasmid Ilc, plasmids are plasmids such as 2 e.g. p3, 8 or pl (
As in S4, "p" is placed in front of the display.

Cells containing plasmids are indicated by identified cells and plasmids in parentheses.

For example, Agrohatatherium chemefaciens (
pTi 15ri) and '802 (pKS 4
K11). Table 1 provides a useful index for the identified plasmids and their relationships. Table 2 provides an index of the deposited bacteria. Figure 1 shows Example 5
Provides a useful comparison of the side buildings mentioned in ゜6 and 8.

The purpose of this example is to teach the expression of a non-T-DNA eukaryotic gene under its own regulation.

↓-1--- position, 4 (Shira slow-V-trans A body Shuri-made restriction site is 1) Digested with Hipdllll filling the 11'1 part of one of its sticky ends with NA polymerase 1, ligated the ends, transformed K802, selected by tetracycline 111I], and from the drug-resistant clones, 1+, 2, and 1 infected.
Removal of restriction sites can also be achieved by characterization with restriction enzymes.

This plasmid is removed from pHR322.
And Kanofu.

1.2 Shuttle Hector's Al-made 11203 was digested in concave I11, and its l''-D
N A Ram 17 fragment (see Figure 2) is Aga L, 2-
After gel electrophoresis, a total of 40% was obtained by discharging and discarding the gel. This fragment was mixed with am IfI linearized p350 and ligated, and the reaction was transformed into 802. Shisosumi 1, which was disrupted from ampicillin-transformed strains, was characterized by restriction mapping, digested with SmaI, and the blunt end:;; was ligated to a l1indIll linker. ``MuyI Ill O'11 Arrival is l1in
After exposure by cleavage with dlll and the flocculent plasmid circularizing itself by ligation, the plasmid was transformed into 802.

The plasmid isolated from the Anpinlin 1 transgenic strain is characterized by the restriction region, and the 414 structure shown in Figure 3, which the plasmid has, is p3.
It was named 95. p376, discussed below, was also isolated at this point. (Figure 3) p395 is digested with Bam If I and its R
The am17T-DNA fragment had its Sma1 site initially converted to Il, but following agarose gel electrophoresis,
Solicited. This Bam17 fragment was mixed and ligated with pRK290 flocnated with -IIBII+. The reaction solution was transformed into 802, and after one selection, the transformed strain was
A 1.5-fold restriction map was used to prepare a plasmid. This particular shisosumi [ha.

p490-8/14.

1376 has its origin in this example, as mentioned above, and now it has been converted to the characteristics of anal muscle III, but there is a deletion of about 0.8 Kbp to the right from there. was found. That Bam as done for p 3 5 above
The Bam II 17F-DN fragment corresponding to 17 was isolated and ligated to pRK290, which had been floccated with 1"II. After transformation selection and plasmid isolation and characterization, the fixed plasmid was designated p45B-1.

1.3 Insertion of the orin q gene in the nonoza l/shiseki ul hair ζ f1 gland The fragment carrying the phaseolin gene was prepared by agarose gel electrophoresis from pKS-KB3.8 that had been digested with llindlll. This fragment was mixed with linearized p490-8/14 in box 1 and ligated.

A kanamycin transformant of K2O2 was used to prepare restriction mapped plasmids from it. Two 114 adults were isolated: p499/
(i/7 had a bean (Shiokyo) sequence to the right of the kanamycin song = 1 (Figure 4) and p499/6/8 had a 9
It was in the opposite direction (Figure 5).

pKS-Kit carrying purified phaseolin gene
The l1indllll fragment of 3.8 is also. The plasmid was mixed with linearized p458-1 and ligated in two rounds.

Again, K8 (31 mu1 was generated from 12 kanamycin hermaphrodite transformants and restriction maps were constructed. Fli and both orientations were isolated: p496-2 (6th [m) and p4
9G-1 (Fig. 5) had phaseolin on the right and left sides of the kanamycin face 1 gene, respectively.

±, 4Ti plus a roundabout [Ll order I cause] The phaseolin and kanamycin genes were integrated into a Ti plasmid maintained in Agrobacterium tumefaciens cells as described in Example 14. Ta.

The two Ti plasmids contain receptors
) was used as: pTi159 is an Off1~pin type plasmid; This is a strain derived from an oc1-pin type plasmid. p499/6/7. p49
9/6/8. pTi159 plasmids containing structures defined by p496-2 and p496-1, respectively, p52
9-8. p52! ]-7, p529-11 and p52
It was named 9-2. Same 4'i in pTi 80
Each model is p539-6. p539-5. p5
They were named 39-2 and p539-1.

”-,-5---11j4! j Tetsu μ and tδ frame p 52
! ] and 1+ 539-series Ti plasmid-containing Agrocycterium J, Nayumefanens cells were stabbed with ↑1 and i! It was used to infect the stems of sunflower plants by injection of 1111 spores of the bacteria.

! -,G--77-(?,!'-'J-7(7)4Q%
ll (del, cction) - The proteinaceous effect of phaseolin was determined by ELIS as described in Example 14.
Detected by As at Kofu (8all). All the nodules examined were found to contain phaseolin; the result was 2 tissues of fresh 0resl+).
ff1i varies between 20 ng and (lnl;
Western plot analysis of SDS-polyacrylamide (F) showed that although it was significantly smaller than original phaseolin, its apparent molecular weight was larger. The exact number and size of Han F varies among hosts.

It is due to the unique post-translational processing of the host.
This is the result of g).

The phaseolin gene, Senger RNΔ, was detected in the gofu as described in Example 12. All examined bumps were found to contain phaseolin (RNA) strands in both poly (Δ) 514 RNA halves;
It is about 0.005% of A. Denatured DNA gel (
Northern Northern
The solution by IJi showed a large molecular weight of 57.1i, the same size as the previous phaseolin mesenzyan jn NA (1,6 Kbp).

Phaseolin again. IE +-I S A LCJ
:-J-(detected in 5 hoot-tissues derived from cells infected by Hector to pTi86).

Phaseolin protein and phaseolin mechanism-RN
Both detected stars of the A signal are modified, and the error encountered when analyzing crown galls transformed by Agrobacterium tumefangconins with unmodified pTi159 and unmodified ρTiA6G. The second is important and substantive.

This example teaches the insertion of a complete phaseolin retrochanter into T-1) N8, which is one imitation of that taught in Example 1. This construction utilizes a valine Ti plasmid, p1'1c5B, which is designed to connect a sequence inserted within the region of the valine synthesis gene.

F↓-14 Ri-yu-hui Hekku's composition Tubarin-type plasmid pTiC58 (8th IPI a
) was digested with Sma I, and the fragment encoding the tuberin synthesis gene was isolated by agarose gel electrophoresis.
It was done. This piece was blunt-end ligated to a B,l I+ linker, which was then exposed by digestion with Bgl I+. The resulting DNA fragment is B
, ] II flocculized pHK290 (Figure 10)
mixed and concatenated. After transformation into K2O2, selected by tetrazycline, plasmid I: i'o,
ff1l[and.

A restriction map was then created. The particular plasmid was named pcI+448 (Figure 8b).

The four C1a1 sites were converted into one Gly'-1 sensitive site by successively recutting pCF44A twice. The plasmid was digested with Ridan-1.

itself was religated and transformed into 802. After selection, identification of the plasmid, and creation of a restriction map, a suitable plasmid lacking the 1 fragment (2 products and 1 site) was digested with a plasmid and regenerated on its own. ligated and transformed into 802.Selection,
After plasmid isolation and restriction mapping, the second deleted plasmid (this time the Rainbow I fragment contains all but the 5° end of the trochanter) was transformed into pMS-noplV.
(Fig. 9, Fig. 8, Fig. 8 C)

2, 2 Kan/beari gene insertion pKS-KB
3.8 (Fig. 11) was digested with H--■, and a 6.0 Kbp fragment containing kanamycin resistance and phaseolin genes was disrupted by agarose gel electrophoresis.

This fragment is a C1a I flocculated pks-nopl
V is mixed, j! ! ! and transformed into 802. Plasmids isolated from transformants 1liJ and ampinlin sensitive to kanamycin and te1-razaiclin were restriction mapped and
The structure shown in b is pKS-noplV-
It was named KI13.8t15. A similar gene sequence located to the left of kanamycin resistance in the phaseolin gene chain was found and named pKs-nopl (83,8113).

2.Transfer to 3Ti plasmid 1 technology (see prior art and Example 14) pTiC5
8 was used to transfer the structure to Tubarin-type Ti plasmid 1. pl(S-noplV-KI13.
T1 plasmids C58-nop K old 13 and pC, respectively, resulting from the crosses of 8113 and 115 with pTiC5B.
The former 15 to 58-nop- were characterized by restriction mapping and analysis.

Bacteria containing either of the two plasmids (these plasmids contain the 5′ terminal sequence of the ditrochanter and the
Fragments of the kanamycin/phaseolin gene (with either orientation present within the 3' flanking sequence of the gene) were used separately to infect the stems of sunflower plants by injecting the bacteria.

Detection of Expression Phaseolin gene expression was detected in klN of sunflower gofu by ELISAs as in Example 13.5.

This example is phaseolin, this is bean j', 14. ps
We will teach you how to manipulate the genes of many seed storage proteins of E., o l -u svulgaris L., and how to insert the hector hephaseolin gene into 1Φ, which is described in various other examples.

L 1 Face tears! Gene Book Book 11-74 Ch
aron 248 8G-PVPh177.4 (or
177.4; The genetic cone of phaseolin in S, M, Sun (Fig. 14) is tilted and llam It I
digested by. 3.8k with phaseolin gene
The bp fragment and its flanking sequences were separated by 'i'-t'+If by agarose gel electrophoresis.

They are linearized in the mouth am111 p]31 n322
(12th M) was combined with /XY and connected. The mixture was transformed into IIBIOI and an ampicillin-resistant and tetrazycline-sensitive colony (c
olonies) was selected. Blasmi l' isolated from these colonies were restriction mapped. Plasmids with +i'TL shown in Figure 13 were selected and AG-pPVPl+3.8 (or p3.8
) was named U. Interlinking of the 111 and Ram If 1 sites inactivates both sites.

Another subclone of 177.4 was constructed by digestion with ucoRI and contained extensive 3' flanking sequences and all but the 5'-most end of the theorin gene.
．． After isolation of a 2 kbp fragment and ampicillin selection of transformants of 11 and 01 transformants, one mutant was restriction mapped. The plasmid inserted in the direction in which the 1 site of pBR322 is adjacent to the 5' end of the phaseolin gene and the end within the 3' untranslated region is 8G-pPVP.
The name I+7.2 was released from NJ. (orp7.2.
Figure 15: Sun et al. and SHBhtom e
L Dan,・same''2).

3.2 Kanamine 7i4 gene extraclonin and isolation pl? Z102 (1?, 8, Jorgenson a
t day, (+979) Mol.

gen, Genet, 匪; 65-72) is a colicin E1 plasmid carrying a copy of the transposon l'n5;
pBl1322, which had been previously linearized with the same two enzymes, was ligated and transformed into K2O2. Plasmids isolated from transformants selected for resistance to both ampicillin and kanamycin were subjected to restriction mapping, and the one with the structure shown in Figure 16 was named pKS-4 (=I'&J
It was done.

3.3 Kanamycin resistance and Tsubaki history ρ3.8 was digested by C1a I and Bam1l+,
And the phaseolin gene and a certain pHl? A 4.2 kb plli piece containing 322 sequences was isolated by agarose gel electrophoresis. This is pKS4 (16th
ρ [311322 (first
kanamynon resistance (neomycin phosphotransferase 11) derived from Fig. 2).

N +) 'l river) C1a of ifl gene trochanter Tn5
1/Ilam II 11 pieces. The mixture was ligated and transformed into 802. 1liii to ampicillin and kanamycin:]
After selection in l:1, plasmids were isolated and control iIJ
71 was created. 1. The child with the J-bend seen in Figure 11. Niichi was named pKS-formerly 13.8.

p7.2 was digested with R1 and Ilam Itl, and a 3.0 kbp 1vi piece with base ζ excluding the 5' end of the phaseolin trochanter was isolated by agarose gel electrophoresis. This is pKst (Figure 16)
Transplantation of Tn5 with iodinated pBR322 (Figure 12) by H7°i) with the derived carnomain gene and 1lindIII/Bam jl 111
J71! mixed with +. The mixture is concatenated and K
2O2 was transformed. After selection of colonies resistant to ampicillin and kanamycin, the plasmid was isolated and restriction mapped. The colony having the structure shown in FIG. 17 was named pKS4-KB3.

In pK344B, phaseolin has lost the 5'-most flanking sequence and all 5' flanking regions of the gene (see Figure 14).

EXTRACTION ↓ This example teaches how to remove indo-II from genetic acetate. This is due to the genetic environment
This is the same as placing cl)N8 in vironment).

5 of 7j of unproccssed
′ and: (° Restriction enzyme section 1 in both exons at the end
+7. It is created by a 1.7 mutation on the 41° or 1 site.

These sites are present in both Fuedenryo-Clean and cl)N. c) I) NΔ containing a missing intron can be removed from the gene clone, and the cDNΔ without the corresponding intron spanning two sites can be removed from the gene clone. J-n's 1v
It can be replaced with an i piece. Manipulation of the sequence is also possible: the gene sequence containing the protein I-1:J can be placed in a cDNA delta environment. b) The internal fragment of the transgenic clone is inserted into the corresponding gap cut in the cDNA Δrijin. This method is similar, but technically more so, as introns often contain a site sensitive to the enzyme chosen to make the converted 1'0'1. Country jff. The difficulties were overcome by careful selection of conditions for partial digestion and 1 h generation of the desired fragments by agarose gel electrophoresis. A further modification of this strategy is to manipulate individual introns within a gene without affecting other introns and exons, and to avoid the presence of undesirable intervening restriction sites within introns as described above. This includes step-by-step swapping of the array.

~4-2-”-nioty+4-wife receiving Q-een l-I-
13.8 was partially and completely digested with Sac, I, and Sac, respectively. A 6.4 kbp fragment containing both the 5° and 3' ends of pBR322 hector and its intron was isolated by agarose gel electrophoresis.
The p1111322 plasmid clone (to cl)N created from 1.8 was partially and completely digested with Sac I and EcoRI, respectively, and contained the entire phaseolin cDNA except for the most 5' and 3' terminal sequences. 33k
bp fragments were isolated by agarose gel electrophoresis. These two fragments are ligated together.

11 was transformed into old O1. After Koshi J-nee's election IR,
Bacterial growth, plasmid '+'Li? As a result of creating a restriction map, it was determined that Blasmi 1 had the desired structure. - This plasmid is p3.8-c I) NΔ (Figure 22)
I was asked by name. Its entire structure is shown in FIG.

4.2 p3.8-2 (Se size (he! Archery use-p3.8-
c I) Note that NΔ can be substituted for gene 1) NA source, such as p3.8, which is used in other examples. And p3.8-cDN when used in this way
A would have a different kind of structure, such as one lacking the Indian I'on. Or you can use this strategy from an already created structure!・Can be used to remove 1 con.

- The purpose of this example is to extract pTi159 and other octopines from the master (bud "shooting" position) of the plasmid.
mr (the root "rooting" position), resulting in a deletion of Ti plasmid 1. This derivative is useful because cells transformed with it are easier to regenerate into complete plants than cells transformed with pTi159, which has an intact TMS and trochanter.

pTi159, which has been deleted immediately, can be modified in two ways: Lms ur inactivation and foreign gene insertion. When these two transforms are set at different positions of "r-1)NΔ, each transform is inserted independently by a different shuttle hector. Each shuttle hector created by the transform is It is.

It requires the use of at least two markers that are selectable in Age II Hatathyrium. In this example, pII R325Fl was added to the usual kanamycin Ml pond.
Since Flora J, phenicol stiffness has been utilized.

5.1 Chloradofenicol It 11 Shoden Child Bean Two-N Structure JLpB 11325 is digested by 1linell,
The smooth end OH,: was linked with the Ill linker. The resulting product was digested and recombined by It ind III to give 5 chloramphenicols and 1
Rini ζiM IJ (named pMS-5 ((J
(Fig. 191). Dirt is 5. It functions as the origin of the anal gyrus 111/tan'-11, which contains the trochanter.

9.2kbp linearized DNA fragment 11 is p2f13Q)1
1 (↓ I11 complete disassembly and Ham 111 partial fat), the single is also 1 (1).

C71m zh transgenic trochanter J “1 from pKS-5”
i'-separated.

mixed with the 9.2 kbp linearized fragment, ligated, and then
chloradofenicol N:il
It was selected based on its gender and named pMS-oct, cam203 (Fig. 20).

pKs-oct, cam203 is pTi15! This is a plasmid clone that can be used to construct many '1'L deletion mutants of J. The right arm of TL and the left side of that right arm contain the resistance gene. The left arm of T L was attached to the left side of the 4th trochanter (Ilindn1 site). For example, if p102 is attached, the deletion is 5,2 kb
It is of length p and contains all of the expansion and tmr. If pl(13 is (4 spicy i), the deletion is 3.2k
The length of bp is too long, and it includes all of the ・part of Hit Me and Lmr. See Figure 2.

pKS-ocL, cam203 was digested with llindllll.

p102 or p103 was digested by anal plate 11], and the 2.2 kbp or 2.0 kbp i'-D N fragment was isolated and linearized pKS-ocL, ca.
m203 and transformed.

pKs-ocL, del, 1+, respectively
(Fig. 2) or pl(S-oct, del,,l
(Figure 22) was isolated. These structures are intersected:
(<, transferred to Agrobacterium tumefaciens by homologous &Il it recombination (11omo-1o
1;ous recombinations) and
Chloramphenicol i! It was selected based on its characteristics. Alternatively, using established methods, the plasmid structure is linearized with 1 Bam II and 3L of CpliK for each pl (linked to the Bgl 11 site of 290) is used.
The +jl construction was inserted in 290.

(Margins below) In this example, the Moxibustion Ti plasmid is mutated by deleting the T-DNA between the 1 site in Lmr and the 1 site in Sma1 in tml. The Ti plasmid that can be modified is pTi159. pTiB6, pTiA
66 and others. This configuration is shown in FIG.

6. Isolation of I Cam gene pKS-5 (Figure 19)
It was more digested than d. The smallest fragments are isolated after separation on an agarose gel as taught in Example 5.

6.2 pBR322 clone of T-DNA with a deletion The right arm of the T-DNA deletion was constructed by inserting a Bgl 11 site into the Sma 1 site of p203 (see Figure 2). p203 was digested by SmaI.

Digested with Bgl II, religated and transformed into K2O2. In another configuration, the Bam1ll linker is
gl It linker and appropriate extension] 11
A partial isolate is isolated. The resulting plasmid was named p203-851 1+ and 1
Digested by ■ and 1Iindl[I. The large Jl 11/1lindlll hector containing fragment was ligated with the chloramphenicol resistant fragment isolated as described in Example 6.1. Chloramphenicol resistance is selected after transformation into 802. The resulting plasmid is named p2f (Figure 23).

6.3 T-DNA - Anal drawing of the clone's left hand and arm■
The site is inserted into the 1lpa I site of p202 by digestion with ExpI and ligation with the 11indlll linker. After exposure of the sticky ends by digestion with l1indnl, a 2 kbp 11-fold I fragment with anal gyral ends is isolated. Zaizai ■ Disassemble the 1lpa l fragment at the end.

Transform into K2O2. After a colony containing the desired fragment is isolated, it is characterized and its plasmid is ρ3e (
Fig. 24).

6.4 Construction of T-DNA Deletion Clones The left arm of the clone was obtained by purifying a 2 kbp fragment of 1Iindl [1 degradation of p3e by elution from an agarose gel after electrophoresis.

p2f is cut with Hindl[I, treated with alkaline phosphatase, mixed with the 2 kbp fragment, ligated, transformed into K2O2, and selected for chloramphenicol resistance. Plasmids were isolated from individual colonies and characterized by restriction plasmid production. A plasmid with two arms in the desired tandem orientation is selected and named pKS-oct, delm. (Fig. 25) pKS-oct and del m were introduced into Agrobacterium tumefaciens by hybridization, resulting in a homologous recombina.
nts) are selected by selection with chloramphenicol. Sunflower and tobacco roots and shoots
ts) is.

Planted and viable tumors are tested for orpin as described in other examples.

Example 7 This example teaches a construction in which tmr and tml are deleted, and is an alternative to Example 6.

7.1 The chloramphenicol resistant construct pBI+325 with a BgI n site is digested with llincII, the blunt ends ligated with Bgl II linkers, digested with ln and religated (Figure 26). , chloramphenicol resistance is selected after transformation of K2O2 or GM33. As a result, the resulting plasmid, pKS-6
serves as the source of the Bgl II/Bcl n fragment containing the ■L gene.

7, 2 Structure of Lmr, tml deletion clone 1'p2
03 is digested by lpaI and S,maI.

After ligating the blunt end with the B,l11 linker, BBI
BglII sticky ends were exposed, religated, and transformed into K2O2. The desired structure is confirmed and designated p2. (Fig. 27) 7.3T-DNA deletion clone (pKS-OCRI
The BgIn fragment with the constitutive trochanter of II a) was isolated from pKS-6 and ligated to p2 cut with Bil I. Chloramphenicol resistance is selected after transformation of 802 to 1. The resulting plasmid was pKS-oct, d
ellla (Figure 28), Example 6□
4.

The purpose of this study is to provide an example of mutation in a louse due to the insertion of a chloramphenicol gene. This gene is pMS-6
It was isolated as a Yen VII/Bcl n fragment from p
B, III of 203 is linked to the 1lpa I site which is converted to a II site.

It is ligated to Bgl II, scraped by Bgl II, and religated.

After transformation of K2O2 colonies were selected and BiI
Searched by restriction mapping for 11 insertion sites (Figure 29).

8.2 Isolation of camii gene pKs-6 is digested with Bgl II and Uly I.

The smallest fragments are isolated by agarose gel electrophoresis.

8.3 Construction of mutated T-DNA clone Example 8
．． The modified p203 from 1 is digested with Bgl II, ligated with the prepared cam gene from Example 8.2, and transformed into 802.

After selection for chloramphenicol resistance, isolation from resistant transformants, and characterization by restriction mapping, the plasmid was pKS-oct, tmr (3.
Figure 0).

Example 9 Regeneration in this example involves carrot tumors stimulated by Ri-based TIP plasmids,
-D.

Chilton et al. (1982) Natu
re 295:432-434.

9.1-1 Nehiko Suzu 1ζζ Carrot discoid tissue is inoculated with about 109 cells per 0.1 mff of water. For each 1.5 cm section of the resulting end, one was cut off and a solid (1 to 1
1.5% agar) Mon1er medium (D, A, Tepf
erand J, C, Tempe (1981) C,
R, 1 lebd, 5 eanc, 8 cad, Sci.

Paris 295:153-156) and grown at 25°C to 27°C in the dark. The medium is free from contamination by cells.

Mo that is transferred every 2-3 weeks and lacks hormones and agar.
The cells are further planted and cultured in n1er medium.

9.2 Regeneration of roots into plants Root tissue cultured as described in Example 9.1 has a
Solid supplemented with netin (0,8% agar) Mon1
placed in er medium. After 4 weeks, the fresh callus tissue is then placed in liquid Mon1er medium lacking hormones.

Shaker (150 rpm) at 22℃~25℃ for 1 month.
) during incubation, the callus is separated into a suspension medium and the embryos differentiate and develop into seedlings when placed in Petri dishes containing hormone-deficient Mon1er medium. These seedlings are grown in a medium and "hardened" by exposing them to air with 9 levels of decreasing humidity before being grown in a greenhouse or garden (
transferred to the soil in field plots).

9.3 Functional t for 1/j of hairy root vector
A Ti-based vector without the mr gene is used in place of the Ri-based vector as described in Examples 9.1 and 9.2. Suitable deletion constructs are described in Examples 6.7 and 8.

Example 1O Regeneration in this example involves tobacco tumors stimulated with a Ti-based<TIP plasmid.

Essentially, 8, Barton et al. (198
3) accomplished as described by Ce1l 32:1033-1043.

10.1 Infected Tobacco Tissues in Crown Gall, C. Braun (195,6
) Cane.

Res, 16:53-56, using a method that utilizes transformed stem fragments.

The stems were surface sterilized with 7% commercially available Chloroxox and 80% ethanol, rinsed with sterilized distilled water,
cn+ sections were cut into agar solid MS medium lacking hormones (T, Murashige and F, skoog
(1962) Physiol, Plant, 15:4
13-491) at the base in a Petri dish. Inoculation is accomplished by inserting a syringe needle into the cut basal surface of the stem and injecting the bacteria. 1 per day for stems at 25°C
Incubate in the light for 6 hours. Growing callus (calli)
) is removed from the upper surface of the stem section and 0.2■/m7! The cells were placed in solid MS medium containing carbenicillin and lacking hormones, and transferred to fresh MS-carbenisoline medium three times over the course of a month to see if there were any cells floating in the medium. tested. Sterile tissue was cultured under culture conditions as described above (25°C, 16 hr, :8 h
r, light:dark) and maintained in solid MS medium without supplements.

10.2 Shape Converted 9-bellied clones are 8, B1nn5 and F, Meins.
(1979) Planta 145: 365-36
obtained from sterile tissue transformed as described by No. 9. Callus (calli) was treated with 0.02 nw/6 naphthalene acetic acid (NAA) at 25°C for 2.3 days.
) is converted into a cell suspension by incubation in liquid MS, shaken at 135 rpm, and filtered through 543 and 213 μm stainless steel mesh. The passed filtrate was concentrated and 0.5% dissolved agar,
2.0■/pNAA, 0.3 kinetin and 0.4
g/j! Dirc.

Approximately 8 x 10 cells in 5 ml of MS medium containing yeast extract
'Blated at a concentration of cells/ml.

Colonies that reached a diameter of approximately one fin were collected at the female tip;
Place and grow on solid MS medium containing 2.0 mg/6 NAA and 0.3 nv//kinetin.

The resulting calli are individually divided and tested for transformed phenotype.

10.3 Regeneration of plants The transformed clones are placed on solid MS medium containing 0.3 μ/β kinetin and cultured as described in Example 10.1. The buds that are forming are 1/1O strength M.
They are rooted by placing them on solid (1,0% agar) medium containing S medium salts, 0.4 mg/jH thiamine, lacking sucrose and hormones, and having a pl+ of 7.0. rooted sapling (plantl)
et) were grown in culture medium.

As stated in Example 9.2,
er) and transferred to soil either in a greenhouse or in a field plot.

10.4 The methods described in Vector Examples 10.1, 10.2 and 10.3 are suitable for T1-based vectors lacking a functional tmr gene. A suitable deletion structure is shown in Example 6゜7.
It is stated in 8. These methods too.

Effective when used with R4-based vectors. The method described for infection of transformed stem fragments in Example 10.1 is often useful for establishing TIP-transformed plant cell lines.

Example 11 Phoseolin is Pl+aseolis 1gari
It is an abundant storage protein (approximately 50% of total seed protein) of S.

Transfer of a functional foseolin gene into alfalfa plants and translation of the foseolin menosenger RNA into the stored foseolin, which facilitates storage protein synthesis into the leaf material,
It has important economic value because it is introduced to be used as livestock feed. Alfalfa is a valuable plant for the transfer and expression of foseolin genes. Because,
It is the receptor for livestock feed.
), it grows quickly, is capable of nitrogen fixation through symbiosis with +7zobium, is susceptible to crown gall infection, and is capable of regenerating into alfalfa plants from a single cell or protoplast. be.

This example teaches the introduction of an expressible foseolin gene into a complete alfalfa plant.

11.1 Shuttle vector 1r' Alpha alpha plants as described hereafter.

It is regenerated from crown gall tissue containing genetically engineered Agrobacterium plasmids. In the first step, we recombined two functional foseolin genes into L.
A "shuttle vector" containing mr- and unfold-T-DNA variants is constructed.

This configuration in turn functions downstream of neomycin bospho I.
-Recombined with the promoter of tubalin synthase that has the structural gene (kanamycin resistance) of transferase (NPT II) (reported byM, -D
, CI+1lton, et Dan, (18January
y 1983) 15th Miami Winter
Symposium; J, L, Marx (1983
) January 1983 ) 15th Miami
Winter Symposium.

reference). This type of configuration is illustrated in Example 1.

11.2 Agrobacterium! The previously introduced "shuttle vector" is then transformed by routine techniques (Example 14) into a strain of Agrobacterium containing a Ti plasmid such as pTi15955.

Grouper terriers containing the recombinant plasmids are selected and co-cultured with alfalfa whose cell walls have been regenerated (Marton et al. (1979)).
Nature 277: 129-131; G, J
, Wullemset al. (1981) Proc.
, Nat, Acad, Sci, LISA 78:43
44-4348; and II, [1, IIorsc
b and 11. T, Fraley (18 January
ary 1983) 15th Miami Wint
er Symposium).

Cells are grown in culture and the resulting callus tissue is tested for the presence of specific m-RNA by Northern blot sorting (Example 12) and for the presence of specific proteins by ELISA test (J. L.

Marx (1983) 5science 21 flood 83
0;R,B,)Iorsch andR,T,Fra
ley (18 January 1983) 15th
(See Miami Winter Symposium)
.

11.3 Regeneration of Plants Alfalfa plants are then treated with A, V, P, Dos
SanLos et al. (1980) Z, Pf.
lanzenphysio+.

99: 261-270; Tj, McCoy and
Bingham, E. T. (1977) Plant
Sci, Letters 10: 59-66; an
d X, A, Walker Dan, (1979) Pl
ant Sci, LetLers 16: 23-30
regenerated from callus tissue in a manner similar to that previously used by These regenerated plants are then propagated by conventional plant growing techniques, forming the basis for new commercial varieties.

8m left (+lI 12 In all examples, RNA was extracted.

It is fractionated and detected through 9th order processing.

12. lRNA extraction This process was performed by Silflow et al. (1981)
Biochemis-try seal: 2725-273
It was a modified version of 1.

An alternative to LiCL precipitation for CsCl centrifugation is Mur
ray et al. (1981) J, Mo1. Evo
I, 1'7: 31-42. The use of 2M lithium chloride plus 2M urea for precipitation is described by Rhodes (1975) J.

Biol, chem, 2 Rainbow 808B-8097.

Tissue was treated with 4% Valor aminosalicylic acid using Polytron or Ground Glass Bomogenizer.

1% tri-isopropylnaphthalene sulfonic acid.

Chilled 50 mM Tris-salt ff1 (p
Homogenized in 4-5 volumes of H8,0). Octatool is used as needed to control foaming. Tris-saturated phenol containing 1% 8-hydroxyquinoline was added to the homogenate, then shaken, emulsified, and 20,000-3,000
Centrifuged at 0g for 15 minutes at 4”c.

The upper layer of water was mixed with chloroform/octatool (24:1
) and centrifuged as above.

The concentrated lithium chloride-urea solutions were then each
The final concentration of M was added and the mixture was left at 20°C for several hours. The RN'A precipitate was then centrifuged off and treated with 2M lithium chloride.
The pellets were washed to disperse them. The precipitate was then washed with 70% ethanol-0.3M sodium acetate and dissolved in enough sterile water to form a clear solution. Ethanol was added to the solution and the mixture was placed on water for 2 hours and then centrifuged to remove the miscellaneous 7M. The RNA precipitate was then regenerated and washed with water or sterile salt. Poly without (U)
Redissolved in buffer.

(Left below) 12.2 Poly(U)/Sephadex chromatography Two poly(U)Sephadex (Trademark: Phar
macia, Inc,, Uppsala, 5ive
den) buffer was used. One is unsalted and 20mM T.
ris, 1mM EDTA and 0.1% SO5. The other is the first buffer with 0.1M sodium chloride added. In A426, to create a good meeting.

Need to make 2x storage buffer. And it is necessary to add salt to the portion. Once the final concentration is reached, the buffer is autoclaved.

Poly (U) Sephadex, Bethesda
Obtained from Research Laboratories.

1 for 100 μg of expected poly(A)RNA
Poly(U) Sephadex of g was used. Poly (U)
Sephadex.

Hydrate in salt-free poly-(U) buffer and load into a jacketed column. Raise the temperature to 60℃.

The column was washed with salt-free buffer until the baseline at 2601- was smooth. Finally, the column is equilibrated with salt-containing poly(U) buffer at 40°C.

Concentration is 500μg/mj! The following RNAs were heated in salt-free buffer at 65°C for 5 minutes. Then cool.

Sodium chloride was added to a concentration of 0.1M.

RNA was then added to the column, which was run at a flow rate of 1 m6/min or less, until the optical density fell to a stable baseline.
move. Then, increase the column temperature to 60 °C and RN
A was eluted with salt-free poly(U) buffer. RNA is typically washed out with three column volumes. The eluted RNA was concentrated with secondary butanol to a volume of 10 m
After adding sodium chloride to make M, double the volume of ethanol is added to precipitate. Dissolve the ethanol precipitate in water.

Add N114-acetate to 0.1M. Then precipitate again with ethanol. Finally, RNΔ is redissolved in sterile water and stored at -70°C.

The method used is that of Thomas (1980) Proc, N.
aL, ^cad.

Sci, USA 77:5201 and lloff
man, et al. (1981) J. Biol, C.
hem, 256:2597.

20mM sodium phosphate (pl+ 6.8-7.0)
A 0.75-1.5% agarose gel was solidified.

If a massed band of macromolecules appeared, 6% or 2.2 M voltaldehyde (using a 36% stock solution) was added and the experiment was repeated. Formaldehyde was added to the agarose after cooling to 65°C. When formaldehyde is added, ethidium bromide makes detection difficult. The running buffer was 10mM sodium phosphate (pn
6.8 to 7.0).

Prior to electrophoresis, RNA was treated with denaturing buffer at a final concentration of 6% formaldehyde, 50% formaldehyde, 20mM sodium phosphate buffer, and 5mM EDTA. RNA was incubated in buffer at 60°C for 10-20 minutes. Incubation was stopped upon addition of stop buffer. 20μl
For the sample, 4μ/50% glycerol,
10mM JEDT^, 5mM sodium phosphate and bromophenol blue were added.

Using flooded electrophoresis. before soaking the gel.

RNA was loaded. It was then placed in the gel for 5 minutes at 125 mA.

Then soak the gel in water and reduce the current to 30m (overnight) or 50mA (6-8 hours). Electrophoresis was performed in a cold room with circulating buffer.

12.4 "Northern" Proto) If the gel was plotted to find specific RNA, it was not stained. However, separated marker lanes were used for staining. Staining was done in 0, 1M sodium acetate, 5μg/mj! Destaining was carried out with bromide bromide in 0.1 M sodium acetate for several hours. Visual recognition was facilitated by treatment with water at 60-70°C for 5-10 minutes before dyeing.

Gels to be plotted were soaked in 10x standard saline citric acid (SSC)-3% formaldehyde for 15 minutes.

If large RNA molecules do not elute from the gel, then add 50 min to nick the RNA before processing.
Treated in mM sodium hydroxide for 10-30 minutes.

If base treatment is used, the gel should be neutralized and soaked in 5SC-formaldehyde before plotting. Transfer of RN, A to nitrocellulose was performed by standard orientation.

Prehybridization was carried out at 42°C for a minimum of 4 hours.
0% formaldehyde, 10% dextran sulfate, 5
x SSC, 5x Denhart, 100 p g/m
e-denatured carrier DNA, 20#g/mj! Poly (
A).

40mM sodium phosphate (pH 6.8-7.0)
, carried out in 0.2% SO3. Hybridization was performed by adding the probe to the same buffer and incubating overnight.

Probes were generally used at a concentration of 5 XIO5 cpm/ml or higher.

After hybridization, nitrocellulose was added to 42
2 x SSC, 25mM sodium phosphate at °C.

Washed multiple times with 5mM EDTA and 2mM sodium octate solution. Finally, 1xs at 64°C for 20 min.
Washed with SC.

If the filter is not dry during autoradiography. If the probe has been removed by extensive washing with 1mM ED T^ at 64°C,
The best results were obtained.

Example 13 "Western" plot (performed to discover the origin after SDS polyacrylamide gel electromigration) consists of 5 essentially R, P, Legocki and D, P, S, V
erma (1981)^nalyt, Biochem,
The procedure was as described in Dandan 385-392.

Micro-E L I S A (enzyme-1i
immuno-sorbant assay
) with 96 wells.
The following steps were performed using an n-2 type plate.

■3.1 Binding of antibody to plate 1 - Coat the wells slowly at a rate of 1:1000.
The cells were coated with an antibody (rabbit anti-phaseolin IgG) diluted to 50%. 37 at 200 μn/well
It was kept warm at ℃ for 2 to 4 hours. Cover the plate with saran wrap. The plates were then washed three times with phosphate buffer solution PBS-Tiyeen. Each wash step was separated by 5 minutes. after that.

1% bovine serum albumin (BS^) was added for washing and left for 20 minutes before being discarded.

Washing was performed 5 times or more 3 times using PBS-Tween.

After cutting the tissue into small pieces, 1gl1
1 tissue/mβ phosphate buffer Salice-Tween-2% polyvinyl, pyrrolidone-40 (PBS-Tween-2
%PVP-40>. All samples were stored in water before and after crushing and before and after generation of the phaseolin standard curve. A standard curve was created with tissue vomogenate. A standard curve was then created with a buffer solution to check the recovery of phaseolin depending on the two tissues. After centrifuging the homogenized sample,
Place 100 μl of each sample into wells and incubate at 4°C.
Overnight.

I left it alone. The same was done twice for each sample to avoid mistakes. The plate was sealed during incubation.

13.3 Enzyme Binding After incubating overnight, discard the antigen and fill the wells with PBS-Tween.
Wash 5 times with 1 5 minute interval between each wash.

The conjugate (rabbit anti-phaseolin IgG alkaline phosphatase conjugate) was added to T'BS-Tween 2% pvp.
(contains 0.2% BSA) diluted 1:3000,
5゜μl was added to each well. Then, it was kept warm at 37°C for 3 to 6 hours. After incubation, discard the conjugate and fill the wells with PBS.
- Wash 5 times with Tween, with a 5 minute interval between each wash.

13.4 Analysis Immediately before starting the analysis, add 5 tablets of p-nitrophenyl phosphate (obtained from Si'gma and kept frozen in the dark) to 10 mn of substrate until one tablet is dissolved. Stir. 200μl chamber? Immediately add solution A to each well. The reaction was carried out for various times (e.g. t = 0.
10.20.40.60.90.120 minutes),
Dynatech Micro-ELISA rea
It was measured using der.

When p-nitrophenyl phosphate (colorless) was hydrolyzed by alkaline phosphatase to inorganic phosphoric acid and p-ditrophenol, p-nitrophenol gave a yellow color to the solution. It could be read spectrometrically at 410 nm. The minimum amount that can be detected is 0
．． It was less than 1 ng.

Example 14 Three-pair crosses were generally performed as described below.

Other variations known to the expert may also be used.

E. coli K2O2 (pRK290-based shuttle vector) '0' (pl? 2013)
and a streptomycin-resistant Agrobacterium tumefaciens strain. pRK2013
transferred to a strain carrying the shuttle vector and created a shuttle vector for transfer into Agrobacterium.

Agrobacterium cells harboring shuttle vector sequences were selected among those growing in medium containing both streptomycin and a drug to which the shuttle vector is resistant (usually kanamycin or chloramphenicol). These cells were isolated from E. coli (pPII
p to Agrobacterium cells by crossing with IJI).
PII IJI has moved. pill IJI and pRK
290-based shuttle vectors cannot co-exist within the same cell for long periods of time. Gentamicin (pP
If the cells were grown in a medium containing IIJI (which has a resistance gene), it was possible to select cells lacking the pRK290 sequence. Only cells resistant to streptomycin and gentamicin and kanamycin or chloramphenicol have the T1 plasmid that has undergone double homologous site recombination with the shuttle vector and thus have the desired configuration.

(Left below) Bright; Full 1! Shi no Jyo, 1) (No change in content) Table 2

[Brief explanation of drawings]

Figure 1 is an explanatory diagram comparing the plasmids of Examples 11, 12 and 14 of the present invention, and Figure 2 is an explanatory diagram comparing plasmids of Examples 11, 12 and 14 of the present invention.
'-Explanatory diagram showing the DNA region, Figure 3 shows p395 and p3
Figure 4 is an explanatory diagram showing the structure of p499/6/7, Figure 5 is an explanatory diagram showing the structure of p499/6/8, and Figure 6 is an explanatory diagram showing the structure of p496-2. Figure 7 is an explanatory diagram showing the structure of p496-1, Figure 8 is an explanatory diagram showing the structure of each plasmid, and Figure 9 is an explanatory diagram showing the structure of p496-1.
FIG. 1O is an explanatory diagram showing the structure of pRK290. FIG. 11 is an explanatory diagram showing the structure of pKS-KB3.8. FIG. 12 is an explanatory diagram showing the structure of pBR322. Figure 13 is an explanatory diagram showing the structure of p3.8, 1st
Figure 4 is an explanatory diagram showing the structure of the phaseolin gene, No. 15
The figure is an explanatory diagram showing the structure of AG-pPν薊2 (p7.2), Fig. 16 is an explanatory diagram showing the structure of pKS-4, Fig. 17 is an explanatory diagram showing the structure of pKSIIKB, and Fig. 18 is an explanatory diagram showing the structure of pKS-4.
．． Figure 19 is an explanatory diagram showing the creation and structure of 8-cDNA, Figure 20 is an explanatory diagram showing the construction of pKS-5, and Figure 20 is pMS-o.
ct, an explanatory diagram showing the creation of cam 203, Fig. 21 is an explanatory diagram showing the structure of pKS-ocL, del II, Fig. 22 is an explanatory diagram showing the structure of pKS-oct, del T, and Fig. 23 is an explanatory diagram showing the structure of pKS-ocL, del II. An explanatory diagram showing the creation method, Figure 24 is an explanatory diagram showing the creation method of p3e, and Figure 25 is an explanatory diagram showing the creation method of pKS-.
Explanatory diagram showing how to create oct, del m, No. 26
The figure is an explanatory diagram showing the method for creating pMS-6, and Figure 27 is pMS-6.
2 is an explanatory diagram showing the method for preparing pMS-oct.
, dellla, FIG. 29 is an explanatory diagram showing the method for converting the 1lpa 1 site of p203 into the Bgl 11 site, and FIG. 30 is pMS-oct, t
An explanatory diagram showing the structure of mr. FIG. 31 is an explanatory diagram showing the structure of p203. 2 Agents Patent Attorney Shusaku Yamamoto ==~he Bam HI FIG, 4 FIG, 7 E-no Ω δ deceased FIG, 9 FIG, 10 FIG, 1l pBR322 FIO, 12 FIG 13 FIG, 21 FIG, 22 H Continuation of the first page of the engineering item [Phase] Int, C1,' Identification code Internal reference number @ Inventor Sherry L, Sura American Association ■ Itom
Tana Drive @ Inventor Dennis W., SA United States Government Rand, Al△ 1 Rice Consin 53714 Madison, Shi010 I Rice Consin 53558 Matsukfur;
Avenue 5611 Procedural amendment (method) % formula % 2. Name of the invention Expression of plant genes 3. Relationship with the person making the amendment Patent applicant address United States of America Koi'rad 80301-22
44 Boulder, Misochel Lane 3375 Name
Agrigienetakes Rereach Corporation Representative Zoll Jay Hansen Nationality United States of America 4 Agent 530 Address Osaka TOf 4-3 Nishitenma, Kita-ku, Osaka 17/5
, Date of amendment order (Despatch 1) July 31, 1980 6, Number of inventions to be increased by amendment - 41i1 of the applicant of 〇 subject - (2) Power of attorney (3) Inventions in the specification Detailed explanation of 8, content of amendment + l) Petition and power of attorney Q, details are separate & IC (2) Tables 1 and 1 from page 102 to page 1 of the specification
As shown in the engraving and attachment of Table 2 on page 07 (no changes to the content)

Claims (1)

[Scope of Claims] 1. It has T-DNA into which a plant gene containing a plant promoter and a plant structural gene is inserted. A DNA shuttle vector in which the promoter is adjacent to the 5' end of a plant structural gene, and the plant structural gene is downstream from the plant promoter in the direction of transcription. 2. The vector according to claim 1, wherein the inserted structural gene of the plant contains an intron. 3. The claim that the inserted plant gene is inserted at a certain site within the tml gene of T-DNA. The vector according to paragraph 1. 4. The vector of claim 1, wherein the plant promoter and the plant gene are derived from different genes and are combined to form a functional hybrid plant gene. 5. The vector according to claim 1, wherein the structural gene of the plant contains c. 6. The T-DNA has been modified, and the modification is tm
2. A vector according to claim 1, comprising a mutation in s or tmr. 7. It has T-DNA into which a plant gene containing a plant promoter and a plant structural gene has been inserted. A bacterial strain comprising and replicating a DNA shuttle vector in which the promoter is adjacent to the 5° end of a plant structural gene, and the plant structural gene is downstream in the direction of transcription from the plant promoter. 8. The bacterial strain according to claim 7, wherein the inserted plant structural gene contains an intron. 9. The bacterial strain according to claim 7, wherein the plant gene is inserted at a certain site within the T-DNA rice gene. 10. The bacterial strain of claim 7, wherein the plant promoter and the plant gene are derived from different genes and are combined to form a functional hybrid plant gene. 11. The bacterial strain according to claim 7, wherein the structural gene of the plant contains cDNA. 12. The bacterial strain according to claim 7, wherein the T-DNA has been modified, and the modification includes a mutation in tmr or tmr. 13. The bacterial strain according to claim 7, comprising Agrobacterium tumefaciens or Agrobacterium rhizogenes. 14. The DNA hector is group p529-2゜p529-8. p539-5. p539-9. p
C5B-nop-KB13 or pC5B-nop-
A bacterial strain according to claim 7 selected from KBl15. 15. Inserting a plant gene having a 1al plant promoter and a plant structural gene into T-DNA, thereby forming a bond between the T-DNA and the plant gene,
the plant promoter is adjacent to the 5' end of the plant structural gene, and the plant structural gene is downstream in the direction of transcription from the plant promoter; (A method for genetically modifying a plant cell wall comprising the step of transferring a plant gene conjugate into a plant cell. - The method according to claim 15, comprising the step of detecting the expression of one of the structural genes of the plant in a plant cell containing the DNA/plant gene combination.17. 18. The method according to claim 15, wherein the plant gene encodes a seed storage protein. 19. The method according to claim 15, wherein the plant gene encodes a seed storage protein. 16. The method of claim 15, wherein the gene coats phaseolin. 20. The T-DNA/plant gene combination is maintained prior to said step (bl) as part of a shuttle vector and is replicated. 21. The method according to claim 15, wherein the plant gene is inserted into the octovin type T-DNA in the rice field "1.6" region. 22. The method of claim 15, wherein the cells are derived from a Dicotyledonaus plant. 23. The method of Claim 15, wherein the Dicotyledonaus plant is a member of the genus Combustidae or Leguminose. The method according to item 22. 24. (Step of inserting a plant gene having an al plant bromocous and a plant structural gene into 1'-DNA, thereby linking the T-DNA and the plant gene. and (b) the plant promoter is adjacent to the 5' end of the plant structural gene, and the plant structural gene is transcriptionally downstream from the plant promoter; A plant, plant tissue or plant cell produced by a method of genetically modifying a plant cell wall comprising the step of transferring a plant gene conjugate into a plant cell. 25. The plant of claim 24, comprising the step of: ) detecting the expression of the structural gene of the plant in a plant cell containing the T-DNA/plant gene combination.Plant tissue or plant cell. 26. The plant of claim 24, wherein said structural gene has one or more introns. A plant tissue or a plant cell. 27. The plant of claim 24, wherein said structural gene encodes a seed storage protein. The plant, plant tissue or plant cell according to claim 24. 28. The plant, plant tissue or plant cell according to claim 24, wherein the plant gene encodes phaseolin. 29. Claim 2, wherein said 'I''-DNA/plant gene combination is maintained and replicated prior to said step fbl as part of a shuttle vector.
The plant, plant tissue or plant cell according to item 4. 30. The plant, plant tissue or plant cell according to claim 24, wherein the plant gene is inserted into the octopine type T-DNA in the Lml or "1.(i" region). 31. The cell. 32. The plant, plant tissue or plant cell according to claim 24, wherein said plant is derived from the Dicotyledonaus plant. 32. Claim 31, wherein said Dichothyrednaus plant is a member of the Composites or Leguminoses. Plants, plant fibers, or plant cells described in . (Margins below)